应用抗体芯片捕获活细胞技术筛选和鉴定CD133阴性胶质瘤干细胞的表面标志物

The cancer stem cell (CSC) hypothesis suggests that neoplastic clones are maintained exclusively by a rare fraction of cells with stem cell properties. Although the existence of CD133 positive cells in human glioblastoma is established, more evidence exists for CSCs in CD133 negative cells. Here we report the development of a colony-forming antibody array that identified human brain tumor initiating cells that initiate tumors in vitro and in vivo. Cells are isolated from human brain tumors and planted on colony-forming antibody array, which has 291 antibodies and more than 1000 binding pots (1 antibody has at least 4 pots). Cells can combine with the antibody pots and continue grow on antibody array. We evaluate the results of binding and the outcome of growing, whether there is a colony or not. The cells, which form the colony in specific antibody pots, are isolated again, and were evaluated using dilution colony forming soft agar array and Transwell plate.Few cells (100-1000) were injected in NOD-SICD ((non-obese diabetic, severe combined immunodeficient) mouse brain. If the injection of as few as hundreds cells causes a tumor, the tumor initiating cells of brain tumor will be identified. Thus, the identification of brain tumor initiating cells provides insights into human brain tumor pathogenesis, giving strong support for the CSC hypothesis as the basis for many other solid tumors, and establishes a previously unidentified cellular target for more effective cancer therapies.

如何筛选和鉴定肿瘤干细胞是目前研究的重点和热点。越来越多的研究显示胶质瘤干细胞除CD133阳性群外，还存在CD133阴性群细胞。本研究应用抗体芯片捕获活细胞技术”colony-forming antibody array”，分离并筛选CD133阴性胶质瘤干细胞。该技术可以根据细胞表面抗原进行高通量筛选，筛选细胞可以持续在抗体芯片上生长，观察其能否形成肿瘤细胞球。对照为传统流式细胞仪筛选细胞的结果。继续对筛选细胞进行自我分裂能力，侵袭能力及耐药性检测。将少量最终筛选的细胞（100-1000个）注射入NOD/SCID小鼠原位胶质瘤模型中进行胶质瘤干细胞的鉴定。通过该研究可以发现除CD133以外的胶质瘤干细胞的分子标志物，为肿瘤干细胞的相关研究提供理论和实践基础。

目的背景：如何筛选和鉴定肿瘤干细胞是目前研究的重点和热点。越来越多的研究显示胶质瘤干细胞除CD133阳性群外，还存在CD133阴性群细胞。.主要研究内容：本研究应用抗体芯片捕获活细胞技术“colony-forming antibody array”，分离并筛选CD133阴性胶质瘤干细胞。该技术可以根据细胞表面抗原进行高通量筛选，筛选细胞可以持续在抗体芯片上生长，观察其能否形成肿瘤细胞球。对照为传统流式细胞仪筛选细胞的结果。继续对筛选细胞进行自我分裂能力，侵袭能力及耐药性检测。将少量最终筛选的细胞（100-1000个）注射入NOD/SCID小鼠原位胶质瘤模型中进行胶质瘤干细胞的鉴定。.重要结果：首先45次抗体芯片结果的分析，应用Matlab软件和Pivot table方法对大量数据进行分析得到的热图。从中可以发现在胶质母细胞瘤中均高表达的分子标记物是CD49C，CD49E，CD63， CD147， CD47。在另外一个实验中，通过手工计数发现可以形成细胞克隆球的分子标记物是CD47，CD147，CD49C，CD49E，CD44等。再根据我们的假设“表达量低但能形成肿瘤球”的原则，初步筛选可能的胶质瘤干细胞标记物为CD9，CD165，CD98，BAT-2 MG, DISIAL GD，以及NrCAM。.关键数据及其科学意义：继续对待选的细胞进行进一步的干细胞实验，我们将重点集中在CD98上。CD98有重链和轻链两种蛋白，通过流式细胞检测在U251和U87细胞中，重链的阳性率为99%以上，轻链中xCT的阳性率为6%左右，LAT1为2%-4%左右。同期对照中的CD133阳性率为2%-5%左右。至此，CD98相关蛋白xCT和LAT1是最有可能成为除CD133以外的胶质瘤干细胞的分子标志物。